RT Journal Article
SR Electronic
T1 Ligand-Supported Purification of the Urotensin-II Receptor
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 639
OP 647
DO 10.1124/mol.110.065151
VO 78
IS 4
A1 Ann T. Du
A1 Döne Onan
A1 Diem T. Dinh
A1 Michael J. Lew
A1 James Ziogas
A1 Marie-Isabel Aguilar
A1 Leonard K. Pattenden
A1 Walter G. Thomas
YR 2010
UL http://molpharm.aspetjournals.org/content/78/4/639.abstract
AB A crucial limitation for structural and biophysical analysis of G protein-coupled receptors (GPCRs) is the inherent challenge of purifying and stabilizing these receptors in an active (agonist-bound) conformation. Peptide ligands, such as the vasoactive, cyclic hormone urotensin-II (U-II), may provide new purification tools, via high affinity, pseudo-irreversible binding suitable for ligand-based affinity purification. We show that the U-II receptor (UT) is resistant to desensitization as a result of low phosphorylation and diminished endocytosis. UT also displays an unusual proclivity to remain active with vasoconstriction sustained despite extensive washout of the ligand. To exploit these properties for ligand-supported purification, we modified the U-II ligand by attaching a biotin moiety and spacer arm to the N terminus, creating a novel affinity ligand (Bio-U-II) to interface with streptavidin media. Bio-U-II bound to UT with pharmacological properties analogous to those of the unmodified U-II ligand (high-affinity, pseudo-irreversible binding). The prebinding of Bio-U-II to UT (before exposure to detergent) facilitated specific capture of UT by stabilizing the receptor structure during solubilization with detergent. Solubilization of UT with the most compatible detergent, n-dodecyl β-d-maltoside, was dependent on the critical micelle concentration, and Gαq/11 protein was copurified with captured Bio-U-II-UT complexes. Furthermore, captured Bio-U-II-UT complexes were resistant to dissociation at elevated temperatures, suggesting that UT is relatively thermostable, making it an ideal candidate for future structural and biophysical studies. This work demonstrates the utility of pseudo-irreversible ligands to support the purification of a GPCR during detergent extraction, resulting in the first successful purification of the UT.